CueO is a multicopper oxidase involved in the homeostasis of Cu in Escherichia coli, and functions as the sole cupric oxidase ever found. Differing from other multicopper oxidases, the substratebinding site of CueO is deeply buried under a methionine-rich helical region including α-helices 5, 6, and 7 that interfere the access of organic substrates. We deleted this region, Pro357-His406 and replaced it with a Gly-Gly linker. Crystal structures of the truncated mutant in the presence and absence of excess Cu(II) indicated that the scaffold of the CueO molecule and the metal binding sites were reserved in comparison with those of CueO. In addition, the high thermostability of the protein molecule and spectroscopic and magnetic properties due to the four Cu centers were also conserved after the truncation. As for functions, the cuprous oxidase activity of the mutant was reduced to ca.10% of that of recombinant CueO owning to the decrease in the affinity of the labile Cu site for Cu (I) ions, although activities for laccase substrates such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, and 2,6-dimethoxyphenol increased due to the changes in accessibilities of these organic substrates towards the type I Cu site. The present engineering of CueO indicates that the methionine-rich α-helices function as a barrier to interfere with the access of bulky organic substrates to provide CueO with the specificity as cuprous oxidase.Keywords: CueO; multicopper oxidase; homeostasis; truncated mutant; X-ray crystal structure A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT3 IntroductionMulticopper oxidases (MCOs) are enzymes containing a multiple copper center to catalyze the oxidation of a variety of substrates such as polyphenols, aromatic polyamines, L-ascorbate, and metal ions concomitantly with the four-electron reduction of dioxygen to water. [1][2][3][4][5] Laccase, the largest subfamily of MCOs, shows multiple functions including lignin degradation, pigmentation, and pathogenesis in fungi as well as lignin biosynthesis and wound healing in plants. [6][7][8][9][10] Therefore, structures and functions of new MCOs such as Escherichia coli CueO (formerly called YacK) [11][12][13] andBacillus subtilis CotA, 14,15 have been discussed in comparison with those of laccase.CueO is a 53.4-kDa periplasmic protein involved in the Cu efflux system, together with CopA, the P-type ATPase. 16 CueO is responsible for the oxidation of cuprous ion to less toxic cupric ion and the oxidation of enterobactin to prevent the copper-catalyzed Fenton reaction so as not to sequester iron from the environment. 17,18 CueO also catalyzes the oxidation of organic compounds including 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), p-phenylenediamine (p-PD), and 2,6-dimethoxyphenol (2,6-DMP). Oxidase activities of CueO toward these substrates are considerably low, but are fairly enhanced in the presence of an excess Cu(II) ions. 11,12 That is, the enzymatic activity of CueO is regulated by Cu ions in ...

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From the photosynthetic green sulfur bacterium Chlorobium tepidum (pro synon. Chlorobaculum tepidum), we have purified three factors indispensable for the thiosulfate-dependent reduction of the small, monoheme cytochrome c 554 . These are homologues of sulfur-oxidizing (Sox) system factors found in various thiosulfate-oxidizing bacteria. The first factor is SoxYZ that serves as the acceptor for the reaction intermediates. The second factor is monomeric SoxB that is proposed to catalyze the hydrolytic cleavage of sulfate from the SoxYZ-bound oxidized product of thiosulfate. The third factor is the trimeric cytochrome c 551 , composed of the monoheme cytochrome SoxA, the monoheme cytochrome SoxX, and the product of the hypothetical open reading frame CT1020. The last three components were expressed separately in Escherichia coli cells and purified to homogeneity. In the presence of the other two Sox factors, the recombinant SoxA and SoxX showed a low but discernible thiosulfate-dependent cytochrome c 554 reduction activity. The further addition of the recombinant CT1020 protein greatly increased the activity, and the total activity was as high as that of the native SoxAX-CT1020 protein complex. The recombinant CT1020 protein participated in the formation of a tight complex with SoxA and SoxX and will be referred to as SAXB (SoxAX binding protein). Homologues of the SAXB gene are found in many strains, comprising roughly about one-third of the thiosulfate-oxidizing bacteria whose sox gene cluster sequences have been deposited so far and ranging over the Chlorobiaciae, Chromatiaceae, Hydrogenophilaceae, Oceanospirillaceae, etc. Each of the deduced SoxA and SoxX proteins of these bacteria constitute groups that are distinct from those found in bacteria that apparently lack SAXB gene homologues.

Bacillus subtilis yumC encodes a novel type of ferredoxin-NADP 1 oxidoreductase (FNR) with a primary sequence and oligomeric conformation distinct from those of previously known FNRs. In this study, the crystal structure of B. subtilis FNR (BsFNR) complexed with NADP 1 has been determined. BsFNR features two distinct binding domains for FAD and NADPH in accordance with its structural similarity to Escherichia coli NADPH-thioredoxin reductase (TdR) and TdR-like protein from Thermus thermophilus HB8 (PDB code: 2ZBW). The deduced mode of NADP 1 binding to the BsFNR molecule is nonproductive in that the nicotinamide and isoalloxazine rings are over 15 Å apart. A unique C-terminal extension, not found in E. coli TdR but in TdR-like protein from T. thermophilus HB8, covers the re-face of the isoalloxazine moiety of FAD. In particular, Tyr50 in the FAD-binding region and His324 in the Cterminal extension stack on the si-and re-faces of the isoalloxazine ring of FAD, respectively. Aromatic residues corresponding to Tyr50 and His324 are also found in the plastid-type FNR superfamily of enzymes, and the residue corresponding to His324 has been reported to be responsible for nucleotide specificity. In contrast to the plastid-type FNRs, replacement of His324 with Phe or Ser had little effect on the specificity or reactivity of BsFNR with NAD(P)H, whereas replacement of Arg190, which interacts with the 2 0 -phosphate of NADP 1 , drastically decreased its affinity toward NADPH. This implies that BsFNR adopts the same nucleotide binding mode as the TdR enzyme family and that aromatic residue on the re-face of FAD is hardly relevant to the nucleotide selectivity.

From Bacillus subtilis cell extracts, ferredoxin-NADP + reductase (FNR) was purified to homogeneity and found to be the yumC gene product by N-terminal amino acid sequencing. YumC is a ~94 kDa homodimeric protein with one molecule of non-covalently bound FAD per subunit. In a diaphorase assay with 2,6-dichlorophenol-indophenol as an electron acceptor, the affinity to NADPH was much higher than to NADH, with K m values of 0.57 vs. >200 µM. K cat values of YumC with NADPH were 22.7 and 35.4 s -1 in diaphorase and in a ferredoxin-dependent NADPH-cytochrome c reduction assay, respectively. The cell extracts contained another diaphorase-active enzyme, the yfkO gene product, but its affinity for ferredoxin was very low. The deduced YumC amino acid sequence has high identity to that of the recently identified Chlorobium tepidum FNR. A genomic database search indicated that there are more than 20 genes encoding proteins that share a high level of amino acid sequence identity with YumC and annotated variously as NADH oxidase, thioredoxin reductase, thioredoxin reductase-like protein, etc. These genes are found notably in Gram-positive bacteria except for Clostridia, and less frequently in archaea and proteobacteria. We propose that YumC and C. tepidum FNR constitute a new group of FNR which should be added to the already established plant type, bacteria type, and mitochondria type FNR groups.

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Reaction center preparations from the green sulfur bacterium Chlorobium tepidum, which contain monoheme cytochrome c, were studied by flash-absorption spectroscopy in the near-UV, visible, and near-infrared regions. The decay kinetics of the photooxidized primary donor P840(+), together with the amount of photooxidized cytochrome c, were analyzed along a series of four flashes spaced by 1 ms: 95% of the P840(+) was reduced by cytochrome c with a t(1/2) of approximately 65 micros after the first flash, 80% with a t(1/2) of approximately 100 micros after the second flash, and 23% with a t(1/2) of approximately 100 micros after the third flash; after the fourth flash, almost no cytochrome c oxidation occurred. The observed rates, the establishment of redox equilibrium after each flash, and the total amount of photooxidizable cytochrome c are consistent with the presence of two equivalent cytochrome c molecules per photooxidizable P840. The data are well fitted assuming a standard free energy change DeltaG degrees of -53 meV for electron transfer from one cytochrome c to P840(+), DeltaG degrees being independent of the oxidation state of the other cytochrome c. These observations support a model with two monoheme cytochromes c which are symmetrically arranged around the reaction center core. From the ratio of menaquinone-7 to the bacteriochlorophyll pigment absorbing at 663 nm, it was estimated that our preparations contain 0.6-1.2 menaquinone-7 molecules per reaction center. However, no transient signal due to menaquinone could be observed between 360 and 450 nm in the time window from 10 ns to 4 micros. No recombination reaction between the primary partners P840(+) and A(0)(-) could be detected under normal conditions. Such a recombination was observed (t(1/2) approximately 19 ns) under highly reducing conditions or after accumulation of three electrons on the acceptor side during a series of flashes, showing that the secondary acceptors can stabilize three electrons. From our data, there is no evidence for involvement of menaquinone in charge separation in the reaction center of green sulfur bacteria.